Malignant Tumor/Cancer
Cancer is a disease involving abnormal cell growth in terms of cell number (proliferation) or in cell size with the potential to invade or spread to other parts of the body (metastasis). Cancer cell proliferation is well known but the mechanism(s) driving metastasis is (are) not well known. Cancer is a disease of genomic alterations: DNA sequence changes, copy number aberrations, chromosomal rearrangements and modification in DNA methylation together drive the development and progression of human malignancies. (The Cancer Genome Atlas Network (TCGA), Nature 2008, 455(23): 1061-68.)
Glioma
A glioma is a type of tumor, which arises from glial cells in the brain or spine. The most common site of gliomas is the brain. Gliomas make up about 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors.
Glial cells, the most abundant cell type in the central nervous system, are supportive cells that surround neurons and provide support for and insulation between them. Unlike neurons, glial cells do not conduct electrical impulses. There are two major classes of glial cells in the central nervous system: astrocytes and oligodendrocytes (Kandel E R, et al., Principles of Neural Science, 4th Ed. McGraw-Hill New York (2000), Ch. 2, pp. 20-21).
Gliomas are named according to the specific type of cell with which they share histological features, but not necessarily from which they originate. The main types of gliomas are: astrocytomas—which share histological features with astrocytes (e.g., glioblastoma multiforme is a malignant astrocytoma and the most common primary brain tumor among adults); oligodendrogliomas—which share histological features with oligodendrocytes; brainstem glioma—a glioma that develops in the brain stem; optic nerve glioma—a glioma that develops in or around the optic nerve; and mixed gliomas, such as oligoastrocytomas, that contain cells that share histological features with oligodendrocytes and astrocytes.
Gliomas are further categorized according to their grade, which is determined by pathologic evaluation of the tumor. Of numerous grading systems in use, the most common is the World Health Organization (WHO) grading system for Glioma, under which tumors are graded from I to IV (Louis D N, et al., Acta Neuropathol, 2007, 114(2):97-109).
Grade I tumors are slow-growing, nonmalignant, and associated with long-term survival (e.g., pilocytic astrocytoma).
Grade II tumors are relatively slow-growing but sometimes recur as higher grade tumors. They can be nonmalignant or malignant (e.g., diffuse astrocytoma).
Grade III tumors are malignant and often recur as higher grade tumors (e.g., anaplastic astrocytoma).
Grade IV tumors reproduce rapidly and are very aggressive malignant tumors (e.g., glioblastoma, giant cell glioblastoma, and gliosarcoma).
Medulloblastoma is the most common type of primary brain tumor occurring in children. The term “medulloblastoma” refers to a series of tumors found in the cerebellum of children. Originally classified as a glioma, medulloblastoma (WHO grade IV tumor) is referred to now as an embryonal tumor. Thought to arise from the malignant transformation of progenitors of the external granular layer of the cerebellum, this tumor accounts for approximately 7-8% of all intracranial tumors and 30% of pediatric brain tumors, and, opposed to glial tumors, is primarily characterized by neuronal differentiation.
Glioblastoma
Glioblastoma multiforme (GBM), a WHO grade IV malignant glioma, classification name “glioblastoma”, is the most common and most aggressive primary brain tumor in adults. GBM arises from glial cells and accounts for 40%-60% of all diffuse astrocytic tumors and 10%-15% of all intracranial neoplastic lesions. The biological characteristics of this tumor are exemplified by prominent proliferation, active invasiveness, and rich angiogenesis. (Nakada, M. et al., Cancers, 2011, 3: 3242-3278). GBM is composed of poorly differentiated neoplastic astrocytes. The presence of microvascular proliferation and/or necrosis is essential for histopathological diagnosis of GBM.
GBM is one of the most aggressive human cancers and is very difficult to treat due to several complicating factors: the tumor cells are very resistant to conventional therapies; the brain is susceptible to damage by conventional therapy; the brain has a very limited capacity to repair itself; and many drugs cannot cross the blood-brain barrier to act on the tumor.
Although treatment can involve radiation, surgery and chemotherapy with temozolomide, which is a methylating agent (P. J. Noughton et al. Clin. Cancer Res. (2000) 6:4110-4118), decades of surgical therapy, radiotherapy, and chemotherapy have failed to drastically change survival for GBM. The medium survival of patients with GBM in clinical trial populations treated with multimodal treatment approaches is approximately 12-15 months, with only 3%-5% of patients surviving longer than 36 months. (McNamara. M. G. et al., Cancers, 2013, 5: 1103-1119).
Based on clinical experience, two subgroups of GBMs have been established, primary glioblastoma and secondary glioblastoma, although these two groups are histologically indistinguishable. Primary glioblastoma, which comprises more than 90% of biopsied or resected cases, arise de novo without antecedent history of low-grade disease, whereas secondary glioblastoma progresses from previously diagnosed low-grade gliomas.
According to a gene-expression-based molecular classification described by The Cancer Genome Atlas (TCGA) Network, Glioblastoma multiforme has four distinct molecular subtypes: Classical, Proneural, Mesenchymal and Neural (TCGA Research Network, Nature, 2008, 455: 1061-1068).
Classical GBM tumors are characterized by abnormal amplification and high levels of epidermal growth factor receptor (EGFR) which is a protein found on the surface of some cells that, when bound by epidermal growth factor, sends signals for the cell to keep growing in number (proliferation). The EGFR abnormalities occur at a much lower rate in the three other GBM subtypes. The TP53 gene codes for tumor protein p53 that normally suppresses tumor growth. TP53 is rarely mutated in classical GBM tumors subtype, but is the most frequently mutated gene in other subtypes of GBM.
Proneural GBM tumors are characterized by alterations of platelet derived growing factor receptor A (PDGFRA) and point mutations in IDH1. The gene IDH1 which encodes isocitrate dehydrogenase-1, when mutated, codes for a protein that can contribute to abnormal cell growth. PDGFRA, which plays an important role in cell proliferation, cell migration, and angiogenesis, was also found to be mutated and expressed in abnormally high amounts. PDGFRA alteration only occurs in Proneural tumors and not in any other subtypes. When PDGFRA is altered, too much of its protein can be produced, leading to uncontrolled tumor growth. The patients of this subtype tend to be younger and to survive longer than in other subtypes.
The Mesenchymal subgroup contains the most frequent number of mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene. Frequent mutations in the PTEN (phosphatase and tensin homolog) and TP53 tumor suppressor genes also occur in the Mesenchymal subgroup. PTEN protein acts as a tumor suppressor, helping regulate the cycle of cell division.
While the Neural subgroup has mutations in many of the same genes as the other groups, the group does not stand out from the others as having significantly higher or lower rates of mutations. The Neural group is characterized by the expression of several markers that are also typical of the brain's normal, noncancerous nerve cells, or neurons, such as NEFL, GABRA1, SYT1 and SLC12A5.
These molecular subtypes of glioblastoma multliforme appear to differ in their clinical courses and therapeutic responses. For example, the different subtypes show varying responses to aggressive chemotherapy and radiotherapy, with a difference of around 50% between the subtypes. It has been suggested that the pathology of each subtype might begin from different types of cells, which might explain the variation in response to therapy. The greatest benefit was seen in the Classical and Mesenchymal subtypes, where intensive therapy has significantly reduced mortality; and there was a suggestion of efficacy in the Neural subtype; but the Proneural subtype was less responsive to intensive therapy including conventional chemotherapy or chemo-radiation therapy. (Verhaak, R G, et al., Cancer Cell, 2010, 17(1):98-110.)
TABLE 1Summary of Four Subtypes of Glioblastoma according to TCGA classification (Bartek, J. Jr., et al., J. Neurol Neurosurg Psychiatry, 2012, 83: 753-760)Phillips et alPro-neuralProliferativeMesenchymalVerhaak et alPro-neuralNeuralClassicalMesenchymalSignatureOlig2/DLL3/SOX2MBP/MALEGFR/AKT2YKL40/CD44MutationsTP53 mutationsChrom 7 gain NFkBPI3KChrom 10 lossNF1PDGFRAPDGRRAClinical featuresNon-responder toClinical outcomeClinical outcomechemotherapyimproved withimproved withtemozolomide/temozolomide/radiationradiation
Conversely, a mesenchymal phenotype is the hallmark of tumor aggressiveness in human malignant glioma. Mesenchymal and Classical subclasses exhibit a worse prognosis compared to Proneural tumors, which may be related to the fact that a subset of Proneural tumors displays mutations in the IDH1 gene as well as the glioma-CpG island methylator phenotype (G-CIMP), both favorable prognostic factors (Verhaak, R G, et al., Cancer Cell, 2010, 17(1):98-110).
Aggressive Proliferation, Active Invasiveness, and Angiogenesis of GBM
The aggressive proliferation, active invasiveness, and angiogenesis of GBM are mainly due to highly deregulated signaling pathways in the tumor.
Proliferative activity with histopathologically detectable mitoses is prominent in almost all GBM cases. Two of the most important proliferation signaling cascades frequently deregulated in glioma are the PI3K/Akt/mTOR and Ras/MEK/MAPK pathways which will be discussed later.
Ubiquitous angiogenesis is an outstanding feature of GBMs. The degree of vascularization is significantly correlated with glioma malignancy, tumor aggressiveness, and clinical prognosis. Pro-angiogenic pathways include a sequence of coordinated events that is initiated by expression of angiogenic factors such as vascular endothelial growth factor (VEGF) with subsequent binding to its cognate receptors on endothelial cells.
GBM is highly invasive. Glioma invasion is a complex process involving (1) detachment from the original site; (2) adhesion to the extracellular matrix (ECM); (3) remodeling of the ECM; and (4) cell migration. Migrating glioma cells tend to move along the vessels, dendrites, and fibers in white matter. These characteristics suggest that GBM possesses specific biological mechanisms that mediate its invasive nature. The highly infiltrative nature of human gliomas recapitulates the migratory behavior of glial progenitors during development of the CNS, suggesting that the activators, receptors, and signaling proteins that contribute to neural crest cell migration may be key players in glioma invasion. Accumulating studies have shown that invasion signaling is induced by several kinds of membrane type protein, such as tyrosine kinase receptor (RTK), integrin, CD44, and G protein-coupled receptor (GPCR), as well as intracellular signaling molecules, including PI3K/Akt, and small GTPases, such as Rac1, cdc42, and RhoA. The CD44 antigen is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration.
EGFR and EphA2 (ephrin type-A receptor 2) RTK are expressed in GBM and are co-localized to the cell surface. EphA2 phosphorylation is dependent on EGFR activity, and EphA2 down-regulation inhibits EGFR phosphorylation, downstream signaling, and EGF-induced cell viability (Ramnarain, D. B.; Cancer Res. 2006, 66, 867-874).
Major Glioma Signaling Pathways
Several major signaling pathways have been associated with Glioma (Nakada, M. et al., Cancers, 2011, 3: 3242-3278).
1. Receptor Tyrosine Kinase Pathway (RTK/PI3K/Akt/mTOR Pathway).
The RTK/P13K/Akt pathway regulates various fundamental cellular processes such as proliferation, growth, apoptosis, and cytoskeletal rearrangement. The pathway involves receptor tyrosine kinases (RTKs), for example, epidermal growth factor receptor (EGFR), platelet derived growing factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR), etc., as well as tumor suppressor protein phosphatase, for example, phosphatase and tension homolog (PTEN), and protein kinases PI3K, Akt, and mTOR. The Receptor Tyrosine Kinase pathway (RTK/PI3K/Akt/mTOR Pathway) is shown in FIG. 1.
EGFR gene amplification is the most frequent alteration (approximately 40%) in GBM. EGFR is a transmembrane glycoprotein member of the ErbB receptor family. In GBM, EGFR is dysregulated through overexpression, which arises because of EGFR gene amplification or activating mutations such as EGFRvIII that lead to ligand-independent signaling. EGFR aberrations have been correlated with a classical subtype of GBM (TCGA Research Network, Nature 455: 1061-68; Verhaak, Roel G. W. et al., Cancer Cell, 2010, 17: 98-110). Although it has been suggested that alterations of EGFR may be correlated with increased aggressiveness of GBM (Nakada, M. et al., Cancers, 2011, 3: 3242-3278), EGFR inhibitors (e.g., Gefinitib, Erlotinib) have not elicited clinical responses in patients with GBMs in clinical trials (Rich, J. N., et al., N. Engl. J. Med. 2004, 351, 1260-1261; Haas-Kogan, D. A. et al., J. Natl. Cancer Inst. 2005, 97, 880-887; van den Bent, M. J. et al., J. Clin. Oncol. 2009, 27, 1268-1274).
Overexpression of platelet-derived growth factor receptor (PDGFR), especially PDGFR-α, and platelet-derived growth factor (PDGF) have been observed in astrocytic tumors of all grades, and their association with malignant progression has been suggested (Nakada, M. et al., Cancers, 2011, 3: 3242-3278). PDGFRA amplification (14%), as well as IDH1 mutation, are major features of the Proneural subtype of GBM according to the TCGA classification (TCGA Research Network, Nature 455: 1061-68; Verhaak, R. G. et al., Cancer Cell, 2010, 17: 98-110). Despite deep association of this molecule with GBM, anti-PDGFR therapy using Imatinib yielded only limited clinical responses (Reardon, D. A., et al., J. Clin. Oncol. 2005, 23, 9359-9368; Reardon, D. A., et al., Br. J. Cancer 2009, 101, 1995-2004).
The PI3Ks are widely expressed lipid kinases that promote diverse biological functions. The binding of PI3Ks and RTKs results in activation of Akt through phosphatidylinositol 3,4,5-triphosphate (PiP3) and 3-phosphoinositide dependent protein kinase-1 (PDK1), which affects multiple fundamental cellular processes including cell survival, proliferation, and motility. According to the integrated genomic classification of GBM, PI3K mutations (15%) are associated with the Proneural subtype (TCGA Research Network, Nature, 2008, 455: 1061-68; Verhaak, R. G. et al., Cancer Cell, 2010, 17: 98-110).
Decreased PTEN activity can activate the RTKs/PI3K/Akt pathway since PTEN negatively regulates the pathway by antagonizing PI3K function. Homozygous deletion or mutation of PTEN is a common genetic feature in GBM (40%), resulting in constitutive activation of the RTKs/PI3K/Akt pathway. PTEN loss is associated with both classical and mesenchymal subtypes of GBM, according to the TOGA study (TCGA Research Network, Nature 455(23): 1061-68).
Akt is an STK (Serine/threonine specific protein kinase) that regulates cell growth, proliferation, and apoptosis. Akt activation has been reported in approximately 80% of human GBMs and correlates with the fact that RTKs/PI3K/Akt signaling is altered in 88% of GBM (TCGA Research Network, Nature 455(23): 1061-68). Oncogenic Akt mutations have not been detected in GBM. Akt inhibitor perifosine is undergoing clinical evaluation in malignant gliomas (Nakada, M. et al., Cancers, 2011, 3: 3242-3278).
2. p14ARF/MDM2/p53 Pathway.
The p53 gene encodes a protein that responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, cell death, cell differentiation, senescence, DNA repair, and neovascularization. Following DNA damage, p53 is activated and induces transcription of genes (such as p21Waf1/Cip1) that function as regulators of cell cycle progression at G1 phase. Mouse double minute 2 homolog (MDM2) oncogene inhibits p53 transcriptional activity by forming a tight complex with the p53 gene, and participates in the degradation of p53. The p14ARF gene codes a protein that directly binds to MDM2 and inhibits MDM2-mediated p53 degradation. In turn p14ARF expression is negatively regulated by p53. Thus, inactivation of p14ARF/MDM2/p53 is caused by altered expression of any of the p53, MDM2, or p14ARF genes. The p53 pathway plays a crucial role in the development of secondary GBMs. The p53 gene is the most commonly mutated p53 pathway gene in glioma; however, molecular abnormalities involving other genes in the pathway have also been described. (Nakada, M. et al., Cancers, 2011, 3: 3242-3278). The p14ARF/MDM2/p53 Pathway is shown in FIG. 2.
3. RB Pathway.
The RB (retinoblastoma tumor suppressor protein) pathway suppresses cell cycle entry and progression, as well as the p53 pathway. The 107-kDa RB1 protein encoded by RB1 (at 13q14) controls progression through G1 into the S-phase of the cell cycle (Serrano, M., et al., Nature, 1993, 366: 704-707). The CDKN2A protein (i.e. p16INK4a which is cyclin-dependent kinase inhibitor 2A) binds to cyclin-dependent kinases 4 (CDK4) and inhibits the CDK4/cyclin D1 complex, thus inhibiting cell cycle transition from G1 to S phase. Thus, alteration of RB1, CDK4, or CDKN2A can cause dysregulation of the G1-S phase transition. However, alteration of only the RB pathway is insufficient to induce tumor formation. EGFR amplification enhances the PI3K pro-growth pathway and is typically associated with CDKN2A deletions. CDKN2A loss is associated with the classical subtype of GBM, according to the TOGA study. (Nakada, M. et al., Cancers, 2011, 3: 3242-3278). The RB pathway is shown in FIG. 2.
4. Ras/MEK/MAPK Pathway.
RAS (Rat sarcoma) proteins act as on/off (RAS-GDP/RAS-GTP) switches controlled by RTKs and neurofibromatosis type 1 tumor suppressor gene (NF-1). Activated RAS (RAS-GTP) then activates serine/threonine kinase RAF. RAF activates mitogen-activated protein kinase kinase (MAPKK), also called MEK, which in turn activates MAPK. MAPK activation results in activation of various transcription factors, such as Elk1, c-myc, Ets, STAT1/3, and PPAR.
The NF-1 tumor suppressor gene encodes neurofibromin, which functions primarily as a RAS negative regulator and plays a role in adenylate cyclase- and Akt-mTOR-mediated pathways. There is increasing evidence that the NF-1 gene is involved in the tumorigenesis of not only NF-1-related, but also sporadically occurring, gliomas. In the TOGA pilot study, NF-1 mutation/homozygous deletions were identified in 18% of GBM. Mesenchymal GBMs, having frequent inactivation of the NF-1 (37%), p53 (32%), and PTEN genes, respond to aggressive chemo-radiation adjuvant therapies. (Nakada, M. et al., Cancers, 2011, 3: 3242-3278). The Ras/MAPK pathway is shown in FIG. 3. A global view of the signaling pathways mentioned above is shown in FIG. 4.
In addition to the signaling pathways mentioned above, other signaling pathways may play a role in GBM initiation, migration, and invasion.
Wnt (Wingless-Related/Mouse Mammary Tumor Virus Integration Family) Signaling Pathways
The proteins encoded by the WNT genes play a role in normal embryonic development. The embryonic processes they control include body axis patterning, cell fate specification, cell proliferation, and cell migration. These processes are necessary for proper formation of important tissues including bone, heart, and muscle. Wnt signaling pathways, which are complex, are aberrantly activated across a vast range of malignancies. Wnt proteins also have been implicated in tumorigenesis, and the inappropriate activation of the Wnt pathway results in the onset of several types of cancer, including breast cancer, prostate cancer, glioblastoma, and others. (Camilli, T. C., Biochem. 2010, Pharmacol. 80(5): 702-711; Polakis P., Curr Opin Genet Dev 2007: 17(1):45-51).
The Wnt signaling pathways are a group of signal transduction pathways of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell. The variety of receptors and ligands involved in Wnt signaling lead to a multitude of diverse signal transduction cascades.
The Wnt family of proteins consists of 19 known human members (Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, Wnt16). These secreted lipid-modified signaling glycoproteins are 350-400 amino acids in length, share 20-85% amino acid identity, and have a conserved pattern of 23-24 cysteine residues. The type of lipid modification that occurs on these proteins is palmitoylation of cysteine in the conserved pattern of 23-24 cysteine residues. Palmitoylation initiates targeting of Wnt protein to the plasma membrane for secretion and allows the Wnt protein to bind its receptor due to the covalent attachment of fatty acids. Following their synthesis, secreted Wnt proteins are modified by glycosylation. In Wnt signaling, these secreted proteins act as ligands to activate the different Wnt pathways via paracrine and autocrine routes.
The Wnt signaling pathways are activated by the binding of a Wnt-protein ligand to a Frizzled (“Fz”) family receptor, which passes the biological signal to the protein Disheveled inside the cell. To date, at least ten members of Frizzled family receptors have been identified, all of which are seven-pass transmembrane proteins characterized by an extracellular N-terminal conserved cysteine-rich domain (CRD) that interacts with Wnts. However, to facilitate Wnt signaling, co-receptors may also be required alongside the interaction between the Wnt protein and Fz receptor. Examples include low density lipoprotein receptor-related protein (Lrp5/6), receptor tyrosine kinase (Ryk), and Ror2.
Interaction of Wnts with their receptors and co-receptors is associated with at least three signaling pathways, namely the canonical Wnt/β-catenin pathway, the non-canonical (or heretical) planar cell polarity (PCP) pathway, and the non-canonical (or heretical) Wnt/Ca2+ pathway. FIG. 5 shows these three representative Wnt signaling pathways. The Fz receptors have the ability to discriminate between different Wnt ligands, and as such, activation of one of these three pathways is dictated by the nature of the ligand/receptor interaction. (Camilli, T. C., Biochem. 2010, Pharmacol. 80(5): 702-711). The canonical Wnt pathway leads to regulation of gene transcription, the noncanonical planar cell polarity pathway regulates the cytoskeleton that is responsible for the shape of the cell, and the noncanonical Wnt/calcium pathway regulates calcium inside the cell. Wnt signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine).
Canonical Wnt Signaling Pathway
The canonical Wnt signaling pathway is a well-established, β-catenin-dependent signaling pathway which involves a key mediator, β-catenin. In the absence of Wnt signaling, β-catenin is phosphorylated by casein kinase 1 (CK1) and glycogen synthase kinase 3 beta (GSK3β) within a “destruction complex” formed by several proteins, including the scaffolding protein Axin and the tumor suppressor gene product APC (Adenomatous Polyposis coli). Phosphorylated β-catenin is then recognized by the ubiquitination machinery and sent for degradation in the proteasome. When Wnts bind to their receptors Fz and Lrp5/6, Lrp5/6 are phosphorylated and Disheveled is activated, which leads to inactivation or disassembly of the β-catenin “destruction complex” such that β-catenin phosphorylation is reduced and β-catenin is stabilized. The stabilized β-catenin then translocates to the nucleus where it regulates downstream gene expression by biding to Lef (Lymphoid enhanced transcription factor) and Tcf (T-cell factor), leading to the transcription of Wnt target genes involved in proliferation and tumor progression. Several members of the pathway can be regulated independently of Wnt signaling. For example, GSK-3β can be inhibited by ILK (Integrin Linked Kinase), and is at the intersection of numerous pathways that might regulate its expression. The Canonical Wnt proteins include Wnt1, Wnt2, Wnt3a, Wnt8a, Wnt8b, Wnt10a, Wnt10b (Jiar C H, J Oral Pathol. Med., 2012, 41(4):332-339).
The Canonical Wnt Pathway in Cancer
The stabilization of β-catenin, lack of degradation and ultimately nuclear accumulation has been linked to poorly differentiated morphology (Endo K, et al., Hum Pathol 2003, 31(5):558-565), high proliferative activity (Inagawa S., et al., Clin Cancer Res 2002, 8(2):450-456), and poor prognosis (Wang C M, et al., Cancer 2001, 92(1):136-145). The fate of β-catenin, namely, its accumulation or degradation, is regulated by numerous proteins, which, if not regulated or expressed appropriately, would account for increased β-catenin expression in cancer. This dysregulation may occur due to mutations in the various members of the signaling pathway, or to epigenetic events. Mutations in Wnts themselves are rare. Mutations affecting downstream targets however, are quite frequent in cancer.
The first described, and perhaps best well known role for Wnt/β-catenin signaling is in colon cancer, where nearly 90% of these tumors harbor mutations that result in β-catenin mutation.
Several Wnt therapeutics that target β-catenin pathways have been the subjects of clinical trials in humans.
Non-Canonical Signaling Pathway
The non-canonical signaling pathway is an umbrella term for all Wnt-activated cellular signaling pathways that do not promote β-catenin-mediated transcription, and numerous such pathways have been identified. Unlike the canonical Wnts, non-canonical pathways are unable to transform mammary epithelial cells and are thought to be involved primarily in cell movement and polarity (Veeman M T, et al., Curr Biol 2003, 13(8):680-685; Kikuchi A, et al., Cancer Sci 2008, 99(2):202-208). There are at least two major non-canonical Wnt pathways, the planar cell polarity (PCP) pathway, and the Wnt/Ca2+ pathway. However, because both involve key molecules such as Wnt5A and ROR2, it is quite difficult to discern the Wnt/PCP and Wnt/Ca2+ pathways in human cancer.
The Wnt/PCP pathway has been best described in development, where it coordinates the polarization of cells along embryonic axes. This involves the activation of STAT3, and JAK/STAT signaling (Miyagi C, et al., J Cell Biol 2004, 166(7):975-981). Wnts that play a role in Wnt/PCP signaling include Wnt5A, Wnt11, and Wnt 7a (Wang Y., Mol Cancer Ther, 2009; 8(8):2103-2109). During Wnt/PCP signaling, Wnt/Fz/Ror2 interactions recruit disheveled (Dsh/Dvl) to the membrane, trigger the recruitment of yang and prickle to the membrane of adjacent cells, and the balance between these regulates polarity. Disheveled-dependent Wnt/PCP signaling then transduces signals via JNK, Jun, Daam, RhoA, Rac, Cdc42 and Profilin, and these have cytoskeletal effects that ultimately control both polarity and motility (Carreira-Barbosa F, et al., Development 2003, 130(17):4037-4046; Takeuchi M, et al., Curr Biol 2003, 13(8):674-679; Qian D, et al., Dev Biol 2007, 306(1):121-133). Since these features (meaning polarity and motility) are critical for tumor progression, Wnt/PCP signaling has been implicated in cancer. (Camilli, T. C., Biochem. 2010, Pharmacol. 80(5): 702-711).
The Wnt/Ca2+ pathway involves the release of intracellular calcium downstream of Wnt signaling. Members of the Wnt family involved in the Wnt/Ca2+ signaling pathway include Wnt5a, Wnt11, and Wnt4, and activation of the Fz receptors by these Wnts was shown to result in the activation of calcium-dependent signaling molecules, such as calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC). These molecules can have a cornucopia of effects on downstream signaling that is often dependent on the cellular context. (Camilli, T. C., Biochem. 2010, Pharmacol. 80(5): 702-711).
More Noncanonical Wnt cascades (pathways) have been suggested including Wnt-RAP1 signaling; Wnt-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling; Wnt-protein kinase A signaling; Wnt-GSK-3-mirotubule signaling; Wnt-atypical protein kinase C (PKC) signaling; Wnt-receptor-like tyrosine kinase signaling; and Wnt-mammalian target of rampamycin signaling. These classifications are not rigid since the pathways overlap and intersect with one another and are evolving. (Semenov, M. V.; Cell 2007, 131: 1378).
Endogenous Wnt Antagonists
Activation of the Wnt pathway is regulated by secreted Wnt inhibitors (Miller J R, et al., Oncogene 1999, 18(55):7860-72). These inhibitors affect the binding of the Wnt ligands to their receptors or co-receptors. These Wnt antagonists include the members of the secreted frizzled related proteins (sFRPs) that bind to Wnt proteins directly, and the members of the Dikkopf (Dkk) family that bind to the Wnt co-receptors LRPs. The transcriptional inaction of sFRPs has been detected in a number of cancers, including colorectal cancer (Suzuki H, et al., 2002, 31(2):141-9). Members of the Dkk family have also been shown to have an inhibitory effect on Wnt signaling (Wu, W, et al., Curr Biol 2000, 10(24):611-1614).
Role and Function of Wnt5a
Wnt5a, in accordance with its different effects in the presence of different receptors, has been shown to have either a tumor suppressive or an oncogenic function, depending on the type of cancer. For example, its expression is down-regulated in colorectal cancer, ductal breast cancer, leukemia, and neuroblastoma. (Blanc, E. et al., Oncol Rep., 2005 14(6):1583-1588). Conversely, Wnt5a was shown to be overexpressed in gastric cancer, pancreatic cancer, non-small cell lung cancer, and prostate cancer. Wnt5a gene expression was found to be increased in more metastatic melanoma cells and increased expression led to increased motility. (Weeraratna A T et al., Cancer Cell, 2002, 1(3): 279-288).
Loss of Wnt5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt5a, 14 peptide fragments and a variety of peptide derivatives have been identified and their ability to mimic the effects of Wnt5a on mammary cell adhesion and impaired migration in a breast cancer cell line have been reported. Foxy-5, a hexapeptide (formyl-Met-Asp-Gly-Cys-Glu-Leu), derived from the 12-amino acid long peptide fragment 175 (Asn-Lys-Thr-Ser-Glu-Gly-Met-Asp-Gly-Cys-Glu-Leu) of Wnt5a, acting as an agonist of Wnt5a, was developed for use in breast cancer. It was reported that the Foxy-5 peptide restored adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, but it did not affect the cellular levels of unphophorylated β-catenin or active JNK. Foxy-5 specifically activates the G-protein-coupled protease-activated receptors 1 and 4. In mammalian cells, the hexapeptide sequence Met-Asp-Gly-Cys-Glu-Leu is present solely in Wnt-5 proteins. N-formylation of the hexapeptide cannot be found in mammalian cells. In vitro analyses revealed that both recombinant Wnt5a and Wnt5a-derived Foxy-5 peptide impaired migration and invasion without affecting apoptosis or proliferation of 4T1 breast cancer cells. In vivo experiments showed that i.p. injections of Foxy-5 inhibited metastasis of inoculated 4T1 breast cancer cells from the mammary fat pad to the lungs and liver by 70% to 90% (Safholm, A, et al., Clin Cancer Res, 2008, 14(20):6556-6563).
In melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches were explored to counteract these effects: inhibition of Wnt5a expression or direct blockage of Wnt5a signaling. Boxy, a hexapeptide (t-butoxycarbonyl-Met-Asp-Gly-Cys-Glu-Leu), modified from Foxy-5, developed for use in melanoma, is a potent, selective antagonist of Wnt5a-mediated migration and invasion of melanoma cells, both of which are essential components of the metastatic process in melanoma (Jenei, V. et al., PNAS, 2009, 106 (46): 19473-19478).
Gene expression profiling has indicated that Wnt5a may be a marker of aggressiveness in melanomas (Bittner M, et al., Nature, 2000, 406:536-540), where Wnt5a overexpression correlates significantly with the survival and the development of metastases.
Although it has been implicated in metastatic processes for non-brain cancers, Wnt5a has not been extensively studied in gliomas.
Brain Cancer (Glioma and Medulloblastoma) and Wnt5a Pathways
Immunohistochemical analyses revealed that Wnt5a expression was higher in human GBM than in normal brain tissue and in low-grade astrocytoma. The overexpression of Wnt5a increased the proliferation of GBM-05 and U87MG cells in vitro. In contrast, the downregulation of Wnt5a expression as the result of RNA interference reduced proliferation of GBM-05 and U87MG cells in vitro, and reduced tumorigenicity of these cells in vivo. The data suggested that Wnt5a signaling is an important regulator in the proliferation of human glioma cells (Yu, J. M., et al., Cancer Lett., 2007, 257(2):172-181).
Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. An independent study of the role of Wnt5a signaling in human glioma has been conducted to unravel the mechanism that stimulates this invasiveness. The results showed that Wnt5a was predominantly and commonly overexpressed among all 19 Wnt families in glioma-derived cell lines; Fz-2, -6, and -7 were dominantly expressed among 10 Fz members in glioma-derived cell lines; and expression of Ror2, a Wnt5a receptor, was very low. These findings suggest that signaling pathways could be activated in glioma cells through overexpression of Wnts or Fz. An immunohistochemical study also revealed high expression of Wnt-5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt-5a expression was correlated with the clinical grade. Knockdown of Wnt-5a expression suppressed migration, invasion and expression of matrix metalloproteinase-2 of glioma cells. Reciprocally, treatment with purified Wnt5a ligand resulted in stimulation of cell migration and invasion. MMP-2 inhibitor suppressed the Wnt5a-dependent invasion of U251 cells (Kamino, M., et al., Cancer Sci, 2011, 102(3): 540-548).
The receptors of Wnt5a that mediate cellular responses of glioma have not been identified. It has been reported that knockdown of receptor-like tyrosine kinase (Ryk), but not of Ror2, suppressed the activity of MMP-2 and Wnt5a-dependent invasive activity in glioma cells. These results suggest that Ryk is important for the Wnt5a-dependent induction of MMP-2 and invasive activity in glioma-derived cells, and that Ryk might have a novel patho-physiological function in adult cancer invasion (Habu, M., et al., J. Biochem, 2014, 156(1): 29-38).
Medulloblastoma (MB), the most common type of primary brain tumor occurring in children, is the most common infratentorial primitive neuroectodermal tumor (PNET) originating in the brain and is a highly malignant primary brain tumor. The canonical signaling pathway is well known in MB. In contrast, very little research about the non-canonical Wnt signaling pathways in MB has been done. Recent studies in MB demonstrate that Wnt5a and Ror2 are additional mechanisms contributing to dysregulation of the non-canonical Wnt signaling pathway, and that Ror2 may play a role as an oncosuppressor (Lee, S. E., et al., Brain Pathology, 2013, 23: 445-453).
In each case, the aforementioned studies targeted Wnt5a signaling pathway in bulk brain glioma tumor cells.
Brain Cancer Stem Cells (or CNS Cancer Stem Cells)
Traditionally, stem cells were thought to be located only in tissues where differentiated cells were most susceptible to loss and the need for replacement great, such as the skin (Huelsken et al., Cell 105: 533-45, 2001), intestinal epithelia (Potten et al., Development 110: 1001-20, 1990) and the blood (Morrison et al., Annu Rev Cell Dev Biol 11: 3-71, 1995). Indeed, the best-known example of an adult stem cell is the hematopoietic stem cell (HSC), which is found in the bone marrow and is ultimately responsible for the generation of al blood cell types throughout the life of the animal (Morrison et al., supra.; Weissman, Cell 100: 157-68, 2000; Weissman, Science 287: 1442-6, 2000). Since the adult central nervous system (CNS) was thought not to exhibit a significant amount of neuronal death, and to have no regenerative capacity, the existence of neural stem cells seemed both unlikely, and unnecessary. However, in 1992 two independent groups successfully demonstrated the existence of precursor cells within the adult mammalian CNS with the ability to give rise to new neurons (Reynolds and Weiss, Science 255: 1707-10, 1992; Richards et al., Proc Natl Acad Sci USA 89: 8591-5, 1992). The source of the new neurons was identified as stem cells that line the entire ventricular neuroaxis of the adult mammalian CNS (Reynolds and Weiss, 1992).
Like stem cells found in other tissues, CNS stem cells (or neural stem cells (NSCs)) have been shown to demonstrate the defining in vitro stem cell characteristics (Hall et al., Development 106: 619-33, 1989; Potten et al, supra.) of proliferation, extensive self-renewal, generation of a large number of progeny, multi-lineage differentiation potential and the in vivo characteristic of regenerating tissue after injury.
One role of stem cells is to divide and give rise to more committed precursor cells with the ability to proliferate and generate a large number of undifferentiated cells. Ultimately, it is the progeny of these more committed precursor cells types that give rise to differentiated progeny. Thus, stem cells can be thought of as a relatively quiescent reservoir of uncommitted cells with the ability to divide throughout the lifespan of the animal and with an extensive proliferation potential, while progenitor cells are more committed and divide more frequently but have more limited proliferation potential over time. Both during development, and in the adult, the proliferation of stem and progenitor cells underpins cell genesis.
The concept of tumors arising from a small population of cells with stem cell characteristics that contribute to the growth and propagation of the tumor is not new to the cancer biology field. The idea was proposed in early 1970's and experimentally confirmed in studies on acute myelogenous leukemia (AML), where low frequency tumor initiating cells were demonstrated to resemble normal hematopoietic stem cells (HSCs). These studies suggested that leukemia stem cells were the direct descendants of HSC or the produce of a more differentiated cell that had acquired HSC features. Discovery of stem cells outside of the blood system raised the possibility that cancers of solid tissues may also contain stem like cells. The existence and isolation of tumor initiating stem-like cells in solid tumors was first demonstrated in human breast cancer tissue, an approach that has also been applied to tumors of the CNS.
Several groups have reported on the ability of cells derived from human glioma tissue to generate neurosphere-like cells in culture, suggesting the presence of NSCs within CNS tumors. It has been demonstrated, based on fluorescence activated cell sorting (FACS) isolation of “side-population” cells, that the well-established glioma cell line U87MG contains a minor population of neurosphere-forming cells that retain in vivo malignancy (C. Hirschmann-Jax, Proc Natl Acad Sci 2004, 101(39):14228-233). Galli and colleagues (Galli et al., Cancer Research (2004) 64: 7011-7021) reported on the isolation, propagation and serial transplantation of tumor neural stem cells (tNSCs) from human glioblastoma multiforme (GBM) that exhibit near identical functional properties as NSCs derived from embryonic and adult CNS. These GBM tNSCs are prominin positive precursors, which display the critical neural stem cell features in vitro, can be expanded in a stable fashion and, throughout serial transplantation-culturing cycles, reproduce the original tumor-initiating characteristics. Together, these studies strongly support the hypothesis that CNS tumors contain a population of stem cells that may be responsible for tumor initiation and malignancy. The tNSCs can be sorted from other GBM cells using FACS by virtue of the expression of CD133 on the tNSCs (Singh et al., Nature (2004) 532:396-401).
The cancer stem cell (CSC) hypothesis suggests that cancers are organized into aberrant cell hierarchies in which “differentiated” daughter cells that have limited capacity to proliferate are produced by a subset of parent CSCs that replicate indefinitely, i.e., only CSCs have the capacity to sustain tumor growth and are responsible for recurrence after therapy fails (Gilbertson, Nature, 2012, 488(7412): 462-463). Until 2012, evidence for the existence of cancer stem cells had been controversial. Drissens (Driessens, G., Nature, 2012, 742: 527-530) and Chen (Chen, J, Nature, 2012, 7412: 522-526) provided elegant evidence to support the existence of CSCs, which offers a sea change in the way we think about and treat cancers.
Cancer Stem Cell Markers
CD133 is considered a marker of stem cells in diverse normal tissues and cancer types. With regard to brain tumors, Singh et al. were the first to describe a CD133 positive tumor cell population, with stem cells characteristics, that is capable of self-renewal and exact recapitulation of the original tumor when transplanted into immunodeficient mouse brains (Singh S K, et al., Nature 2004, 432: 396-401; Singh S K, et al., Cancer Res 2003, 63:5821-5828). Other putative markers of GCSCs include L1CAM, CD44, CD15, Integrin a6 (Brescia P., J Carcinogene Mutagene 2011, 51), and EphA2 (Binda E., et al., Cancer Cell 2012, 22(6): 765-780). The neuronal cell adhesion molecule L1CAM (L1, CD171) is required for maintaining the growth and survival of CD 133 positive glioma cells with stem-like properties. Several reports have shown the utility of the cell surface marker CD44 in the identification of cancer stem cells in different type of tumors, including one example of the use of CD44 as a stem cell marker in glioblastoma (Anido J, et al., Cancer Cell 2010, 18:656-668). CD15 is a cell surface protein selectively expressed in cells with tumor initiation capacity. Integrin a6, important for the interaction with laminin expressing endothelial cells in the microenvironment, is a component of the extracellular matrix whose contact is important for glioma stem cells maintenance. The integrin—α6-laminin interaction has been reported to play an important role in the subventricular zone (SVZ) of the lateral ventricles in the adult brain. EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs and drives self-renewal and tumorigenicity in hGBM TPCs (Binda E., et al., Cancer Cell 2012, 22(6):765-780).
Gliomas Cancer Stem Cells (GCSC) Targeting Treatment
GCSCs are distinguished by the ability to self-renew, the ability to initiate brain tumors, the expression of neural stem cell markers, and multipotency, which is the capacity to differentiate into cells with a neuronal, astrocytic, or oligodendroglial phenotype. GCSCs express antigens specific for neural stem and progenitor cells: Nestin, CD133 (prominin-1), Musashi-1, and Bmi-1. Sonic hedgehog homolog (SHH) and Notch are key regulators of neural progenitors and have been found to be altered or overexpressed in GCSCs (Nakada, M. et al., Cancers, 2011, 3: 3242-3278). FIG. 6 shows Gliomas cancer stem cell pathways.
GCSCs are radioresistant and chemoresistant, which eventually results in tumor recurrence. Targeting GCSCs for treatment is critical. Five general methods have been proposed for targeting the GSCs: (1) to develop new therapeutic agents to target signaling pathways of CSCs; (2) to use a radio-sensitizer to enhance the radiotherapy effect on CSCs; (3) to use immune cells to attack the CSCs: (4) to use a differentiation agent to promote the CSCs to differentiate into normal cells; and (5) gene therapy (Cho, et al., Cell Transplant. 2013, 22(4):731-9).
Therapeutic agents have been used to target signal pathways to treat GBM, include Wnt pathways, sonic hedgehog (shh), Notch, homeobox (HOx) family, B-lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), PTEN, telomerase, efflux transporters, EGF, micro-RNA, and VEGF receptors (Cho, et al., Cell Transplant. 2013, 22(4):731-9).
Canonical Wnt-signalling activates the translocation of β-catenin to the nucleus, where it acts as a transcription factor of specific target genes, and Wnt-β-catenin signaling has proven roles in both normal stem cells and GCSCs. Wnt-β-catenin signaling can contribute to radio-resistance in GCSCs and may be a therapeutic target for GCSCs in brain tumors, as this pathway supports motility/invasiveness of gliomas and drives changes resembling epithelial to mesenchymal transition. (Cruceru, M. L., et al., J. of Cellular & Molecular Medicine, 2013, 17(10): 1218-1235).
To date, the pathways/mechanism behind the migration, invasion, and metastasis of brain gliomas are still not understood. It is now accepted that cancer stem cells are responsible for resistance to chemo- and radio-therapy and re-occurrence of tumor cells, but to date, no research targeting the Wnt5a non-canonical signaling pathways to reduce invasiveness of glioma cancer stem cells has been reported, and no therapeutic agent that specifically targets Wnt5a non-canonical signaling pathway in brain cancer stem cells has been identified. The described invention addresses these problems and provides peptide derivatives of Wnt5a that reduce invasiveness of glioma cancer stem cells, at least in part by directly or indirectly affecting Wnt5a signaling.